The Aurora kinases are a family of serine/threonine kinases. Aurora A and B kinase are associated with mitotic events of cell cycle, while Aurora C kinase is expressed only in testis (Sasai K, Katayama H, Stenoien D L, Fujii S, Honda R, Kimura M, et al, Aurora-C kinase is a novel chromosomal passenger protein that can complement Aurora-B kinase function in mitotic cells. Cell Motil. Cytoskeleton, 2004; 59:249-263). Aurora-A kinases, also called Aurora-2, STK6, ARK1 and Aurora/IPL-1 related kinase, associates with centrosomes and microtubules during mitosis. Aurora A kinase (hereinafter “Aurora A”) localizes to centrosomes and regulates the association between cell cycle machinery and centrosomes (Hirota T, Kunitoku N, Sasayama T, Marumoto T, Zhang D, Nitta M. Aurora-A and an Interacting Activator, the LIM Protein Ajuba, Are Required for Mitotic Commitment in Human Cells. Cell 2003; 114: 585-598; Dutertre S, Cazales M, Quaranta M, Froment, C, Trabut, V, Dozier, C. et al. Phosphorylation of CDC25B by Aurora-A at the centrosome contributes to the G2-M transition. J. Cell. Sci. 2004; 117: 2523-2531).
Aurora B kinases, also known as Aurorakinase B, Aurora B, Aurora-1, and hereinafter “AurkB”, localizes to the kinetochores from prophase to metaphase and to the central spindle and the midbody in cytokinesis (Carmena M. and Earnshaw W C. The cellular geography of aurora Kinases. Nat. Rev. Mol. Cell Biol 2003; 4: 842-854). AurkB associates with chromosome passenger proteins, inner centromere protein (INCENP), Survivin and Borealin protein to form a quaternary chromosome passenger complex which, along with its sub complexes (AurkB and INCENP) are thought to be required for spindle check point, cytokinesis and phosphorylation of Histone H3, respectively. (Schumacher J M, Golden A, and Donovan P. AIR-2: An Aurora/Ip11-related protein Kinase associated with chromosomes and midbody microtubules is required for polar body extrusion and cytokinesis in Caneorhabditis elegans embryos. J. Cell. Biol. 1998; 143: 1635-1646; Terada Y, Tatsuka M, Suzuki F, Yasuda Y, Fujita S, and Otsu M. AIM-1: a mammalian midbody-associated protein required for cytokinesis. EMBO J. 1998; 17: 667-676; Giet R and Glover D M. Drosophilia aurora B Kinase is required for histone H3 phosphorylation and condensing recruitment during chromosome condensation and to organize the central spindle during cytokinasis. J. Cell Biol 2001; 152: 669-682). AurkB phosphorylates MCAK and thus plays a vital role in regulation of bi-orientation during mitosis (Giet R, Petretti C and Prigent C. Aurora Kinases, aneuploidy and cancer, a coincidence or a real link? Trends. Cell Biol. 2005; 5: 241-250).
Any discrepancy in functioning of Aurora kinases can lead to mitotic catastrophe resulting in anueploidy or polyploidy, a hallmark of cancer. Indeed, chromosomal instability cause cancer and is clearly associated with cancer evolution and thus resistance to treatment (Duesberg P et al., “The chromosomal basis of cancer” Cell Oncol. 2005; 27(5-6):293-318; Duesberg P et al., “Cancer drug resistance: The central role of the karyotype.” Drug Resist Updat. 2007 Mar. 26). Aurora kinases have been linked to the chromosomal instability. In addition, malfunctions of Aurora kinases are found in a number of cancers, such as non-small cell lung cancer, epidermal, prostate, colon, pancreatic, ovary, breast and oral cancers including all head and neck cancers. (Keen N. and Taylor S. Aurora-kinase inhibitors as anticancer agents. Nature Rev 2004; 4: 927-936). Inhibition of AurkB leads to improper segregation of sister chromatids and failure of cytokinesis. Over-expression of AurkB has been linked to cell proliferation and development of aggressive tumors leading to malignancy. (Ota T, Suto S, Katayama H, Han Z, Suzuki F, Maeda M et al. Increased mitotic phosphorylation of histone H3 attributable to AIM-1/Aurora-B over expression contributes to chromosome number instability. Cancer Res 2002; 62: 5168-5177; Vischioni B, Oudejans J J, Vos W, Rodriguez J A and Giaccone G. Frequent overexpression of aurora B Kinase, a novel drug target, in non-small cell lung carcinoma patients. Mol. Cancer Ther. 2006; 5: 2905-2913; Smith S L, Bowers N L, Betticher D C, Gautschi O, Ratschiller D, Hoban P R. Overexpression of aurora B Kinase (AURKB) in primary non-small cell lung carcinoma is frequent, generally driven from one allele, and correlates with the level of genetic instability. Br. J. Cancer 2005; 19:719-29). Studies so far have not indicated whether the malfunctioning of Aurora is a cause or consequence of cancers. AurkB is intimately involved in preventing chromosomal instability. (Liu Q, Kaneko S, Yang L, Feldman R I, Nicosia S V, Chen J et al. Aurora-A Abrogation of p53 DNA binding and transactivation activity by phosphorylation of serine 215. J Biol Chem. 2004; 279: 52175-52182; Katayama H, Sasai K, Kawai H, Yuan Z, Bondaruk J et al. BRCAI phosphorylation by aurora kinase A induces Mdm2-mediated destabilization and inhibition of p53. Nat. Genetics 2004; 36: 55-62; Ouchi M, Fujiuchi N, Sasai K, Katayama H, Minamishima Y A et al. BRCAI phosphorylation by Aurora-A in the regulation of G2 to M transition. J Biol. Chem 2004; 279:19643-19648). Survivin, part of AurkB complex, is a key protector against apoptosis and/or mitotic catastrophe (Andrews P D. Aurora Kinases: shining lights on the therapeutic horizon? Oncogene 2005; 24: 5005-5015). AurkB therefore appears to have a direct role in tumorigenesis.
Aurora inhibitors have been reported several authors (Ditchfield C, Johnson V L, Tighe A, Ellston R, Haworth C, Johnson T et al. Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2 and Cenp-E to kinetochores. J Cell Biol 2004; 161: 267-280; Hauf S, Cole R W, Terra S, Zimmer C, Schnapp G, Walter Ret al. The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint. J Cell Biol 2003; 161: 281-294 and Harrington E A, Bebbington D, Moore J, Rasmussen R K, Ajose-Adeogun A O, Nakayama, T. VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat. Med 2004; 10:262-267). These inhibitors include ZM447439 (by AstraZeneca) which shows inhibition of 13 other kinases apart from Aurora. AZD 1152 (AstraZeneca) has been shown to inhibit spindle aggregation at the time of mitosis and is being evaluated to determine its effect as a specific inhibitor of Aurora kinase and its impact on cell division in proliferating tumors. Hesperadin (by Boehringer Ingelheim) inhibits AurkB and 25 other kinases, but not Aurora A or C, and causes only polyploidy with no apparent loss to cell viability.
VX-680 (Vertex) inhibits all three Aurora and, in a panel of cancer cells, was found to inhibit proliferation, increase apoptosis induction, and induce tumor cell death. Merck is presently conducting three clinical studies of VX-680 in patients with hematologic cancers, recurrent or non-responsive solid tumors, or cancers for which standard therapy does not currently exist. In these studies, the safety and tolerability of VX-680 are being evaluated when administered as either a 24-hour continuous infusion or as a 5-day continuous infusion. Of the Aurora inhibitors in clinical trials, only VX-680 is highly specific for Aurora, but is associated with toxic bone marrow side effects, and the mechanism of tumor cell death is not completely understood (Giet R, Petretti C and Prigent C. Aurora Kinases, aneuploidy and cancer, a coincidence or a real link? Trends. Cell Biol 2005; 5: 241-250).
Additional Aurora inhibitors have been described. Japanese patent application JP 2005-278472 describes a peptide that inhibits activities of AurkB in the inner centromere protein, specifically bonding to the Aurora protein; and also describes a medicinal composition, screening methods, and kits. Japanese patent application JP 2005-320351 describes a process for preparing tri- and tetra-substituted pyrimidines, the use in preparing Aurora kinase inhibitors, and methods of treatment. US patent application 20050267065 describes compositions and methods for modulating the expression of AurkB, including using chemically modified nucleotides, including small interfering nucleotides. The use of chemically modified nucleotides poses a potential risk of unwanted side effects.
The epidermal growth factor receptor (EGFR, also known as ErbB-1, or HER1 in humans) is a protein tyrosine kinase. Activation of EGFR leads to transmission of proliferative signals to the nucleus and, via activation of transcription factors, leads to increased proliferation, increased migration, increased adhesion, increased angiogenesis and inhibition of programmed cell death pathways (Bundy, L., Wells, S., Sealy, L. “C/EBPbeta-2 confers EGF-independent growth and disrupts the normal acinar architecture of human mammary epithelial cells,” Mol. Cancer. 2005; 4: 43). EGFR is over-expressed in a number of cancers such as head and neck squamous cell carcinoma (HNSCC), non-small cell lung carcinoma (NSCLC), prostate, gastric, epidermal and skin cancers. EGFR is expressed constitutively in many highly aggressive tumors (Rusch V, Mendelsohn J, Dmitrovsky E. “The epidermal growth factor receptor and its ligands as therapeutic targets in human tumors.” Cytokine Growth Factor Rev 1996; 7: 133-41; Salomon D S, Brandt R, Ciardiello F, Normanno N. “Epidermal growth factor related peptides and their receptors in human malignancies.” Crit Rev Oncol Hematol 1995; 19: 183-232) and tumors showing over expression of EGFR are often found to be resistant to chemotherapeutic drugs (James H. Doroshow “Targeting EGFR in Non-Small Lung cancer.” N. Engl. J. Med. 2005; 353(2):200-2002).
EGFR inhibitors such as cetuximab, erbolitin, etc, are known. To enhance the tumoricidal affects of EGFR inhibitors, therapeutic treatments often include use of cytotoxic chemotherapeutic drugs such as cisplatin, foldfox, etc. However, in randomized clinical trials EGFR inhibitors when combined with cytotoxic chemotherapy, no advantage was demonstrated over standard chemotherapy alone (Sui G., Soohoo C., el Affar B., Gay F., Shi Y., Forrester W. C. & Shi Y. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl Acad. Sci. USA 2002; 99, 5515-5520). Accordingly, there is also a need for improved EGFR inhibitors.
Chemically synthesized short nucleic acid molecules, such as siNAs, can specifically and effectively direct homology-specific post transcriptional gene silencing, and therefore may be used as highly effective, selective and potent therapeutics, with minimal side effects. A molecule that specifically inhibits AurkB can block the mechanics of cell division, and therefore is very useful in combination therapy. Similarly, siNA may be used to block EGFR expression.
The present invention provides potent short nucleic acid molecules without any chemical modification having high stability and specificity for AurkB and or EGFR, and are useful as therapeutics alone, or in combination with other therapies for cancer.